3 Top Tips for RNA Extraction From Mouse Brain Tissue

Ribonucleic Acid (RNA) is essential for genetic regulation and expression. That’s why its extraction is a major part of gene expression analyses. In many neuroscience studies, animal models such as mice are used. Accordingly, you may find that you must extract RNA from mouse brain tissue at some point or other. While the extraction procedure itself isn’t complicated, two challenges arise when trying to extract RNA from mouse brain samples. The first challenge is that working with RNA is tougher than working with DNA because RNA is inherently less stable due to its chemical structure. It’s unstable at higher temperatures (> 65°C) and can be degraded in the presence of metal ions or in alkaline conditions. Additionally, RNases are more ubiquitous in standard laboratory conditions than DNases. Even the slightest exposure to RNase can impact RNA stability. RNases are problematic because they are resistant to high temperatures and chemical treatment. The second challenge faced when extracting RNA from mouse brain is the nature of the tissue. In general, isolating intact total RNA is more difficult when processing certain problematic tissues, such as lipid and protein-rich tissues and fibrous tissues. The brain falls under this category due to its high lipid content. This guide outlines the main precautions you should take to overcome these challenges. It’s meant to supplement your main extraction lab manual. If you would like to read further on the topic, please check references 3 and 4.

Lab preparation

Before beginning your work, your lab should be properly prepared to work with RNA. As mentioned before, RNA can be quite fragile if mishandled and RNases are notoriously difficult to remove. There are four factors to pay attention to when preparing your lab: pH, temperature, metal ions, and RNases.

1. Designate one area for RNA extraction: This becomes especially useful if you’re working in a shared lab. Having just one part of the lab designated to RNA extraction and handling limits sources of RNase contamination.

2. Use disposable RNase free plastic ware: The label on the plastic-ware must explicitly indicate that it’s “RNase-free.” Don’t assume that something is RNase-free because it’s sterile as sterilization doesn’t get rid of RNases. In addition, the pipette tips used should contain a filter to prevent any contamination. (Note: If you would like to use glassware, it should be baked at +180°C to +200°C for at least 4 hours. Autoclaving will not get rid of RNases.)

3. Treat reusable plastic ware with EDTA: If you intend to use reusable plastic ware, it must be soaked (2 hours, +37°C) in 0.1 M NaOH/1 mM EDTA (or absolute ethanol with 1% SDS) to get rid of any metal ions. Then rinse with DEPC-treated water and heat to +100°C for 15 minutes.

4. Gather all your lab equipment: Make sure that all the equipment you will be using are available and easy to access. You don’t want to end up scrambling for a pipette mid-extraction.

5. Clean the lab and equipment: Make sure to clean all benches, pipettes etc. in the RNA extraction area using 100% Ethanol or a commercial RNase inactivating agent. Also, reserve all the chemicals used in RNA extraction and analysis for use in RNA applications only.

6. Always keep the temperature low: All RNA samples should be kept at 0 - 4°C while working with them. That’s why it’s important to ensure that you always have ice on hand before collecting samples or removing them from storage.

Sample collection and handling

1. Always wear protective clothing: Gloves, a lab coat, and safety glasses should always be worn. While this is standard lab procedure, it becomes more critical when extracting RNA to preserve the integrity of your sample and for your safety (especially when using organic extraction). A major source of RNase contamination is human skin, so make sure to wear gloves the entire time you are working on your extraction. In addition, don’t touch anything outside the RNA extraction area to prevent RNase transfer.

2. Sample collection: You should make sure that you collect your samples rapidly and that the tissue is treated promptly either by flash freezing, RNAlater, or homogenization buffer to prevent the activation of endogenous RNases.

3. Tissue dissection: If certain parts of the brain such as the striatum or hippocampus must be isolated, dissection must be done quickly on a chilled RNase-free surface. If the brain tissue is left too long (>5 min.) or its temperature rises, the risk of RNA degradation by endogenous RNase increases.

4. Homogenization:

• Before homogenization, make sure that your homogenizer is clean from all fatty residues from previous use. In addition, make sure all its parts are RNase-free, treating each part according to its material (See points 2, 3, and 5 in Lab preparation.)
• Make sure that your tissue is thoroughly homogenized. The homogenization buffer should become turbid with no visible tissue parts suspended.
• As brain tissue is high in fat content, you must take an extra step to remove the fatty material from your sample. After homogenization, centrifuge your sample at 12,000 x g for 10 minutes at 4°C.

A pellet will form at the bottom of the tube containing extracellular material, high molecular weight DNA, and polysaccharides, while a layer of fat collects above the supernatant. Discard the fatty layer and collect the supernatant to continue extraction according to the directions of your extraction kit.

5. Storage:

• In tissue: Tissue should be flash frozen in liquid Nitrogen and kept at -80°C. Never let the brain tissue thaw, even when extracting as this may lead to degradation of your RNA. Commercial storage media are also available. For example, brain tissue can be stored in RNAlater at -20°C.
• Extracted RNA: RNA should be stored at -80°C. If you wish to store RNA for a long duration (years), it should be stored in Ethanol aliquots at -20°C or -80°C.

Troubleshooting

Problem	Possible reason	Action to take
Low yield	Incomplete homogenization	Use a smaller amount of tissue and mince it to make sure it’s completely immersed in the homogenization buffer.
Degraded RNA	Improper handling	As degraded RNA cannot be used, you will have to do another extraction. Pay particular attention to temperature and any RNase contamination.
Contaminated RNA	Improper phase separation	Treat with DNase to remove residual genomic DNA. Purify using column purification.

References

1. Chomczynski, P., & Sacchi, N. (n.d.). Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction, 4.
2. Johnson, S. A., Morgan, D. G., & Finch, C. E. (1986). Extensive postmortem stability of RNA from rat and human brain. Journal of Neuroscience Research, 16(1), 267–280. https://doi.org/10.1002/jnr.490160123
3. Jr, R. E. F. (2009). RNA Methodologies: Laboratory Guide for Isolation and Characterization. Academic Press.
4. Nielsen, H. (2011). Working with RNA. Methods in Molecular Biology (Clifton, N.J.), 703, 15–28. https://doi. org/10.1007/978-1-59745-248-9_2
5. trizol_reagent.pdf. (n.d.). Retrieved from http://tools.thermofisher.com/content/sfs/manuals/trizol_ reagent.pdf